Antimicrobials and methods of use thereof for wound healing

ABSTRACT

This disclosure provides generally for antimicrobial compositions and methods of use comprising an anthocyanin, an anthocyanidin or metabolites thereof. Methods for promoting healing of a wound using these compositions are also disclosed. These compositions have broad spectrum antimicrobial activity and are safe for human and animal uses. Further, these compositions are safe for medical uses and industrial uses as antiseptic preparations to reduce or prevent microbial growth.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a division of U.S. application Ser. No. 15/189,510,filed Jun. 22, 2016, which is a division of U.S. application Ser. No.14/264,553, filed Apr. 29, 2014, now U.S. Pat. No. 9,498,413, issuedNov. 22, 2016, which claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application No. 61/818,275, filed May 1, 2013, thedisclosures of which are incorporated, in their entirety, by thisreference.

FIELD OF THE INVENTION

The present invention generally relates to antimicrobial compositionsand methods for promoting healing of a wound and more specifically tomethods and compositions including the administration of an anthocyaninor an anthocyanidin or metabolites thereof for promoting wound healingby reducing or preventing microbial growth and inducing the activationof growth hormones. Pharmaceutical and nutraceutical compositionscontaining anthocyanin or anthocyanidins or metabolites thereof suitablefor administration to a mammal for promoting or inducing wound healingare also described.

BACKGROUND OF THE INVENTION

There are many illnesses and conditions which are effectively treated bythe application of suitable antimicrobial agents. Many microorganisms,however, are increasingly difficult to treat because of resistance orallergic reactions to current antimicrobial agents. The development ofresistance is due in part to overuse of the antibiotic and subsequentbacteria mutation. (Blaser, M. Antibiotic overuse: Stop the killing ofbeneficial bacteria Nature 476, 393-394 (25 Aug. 2011).

The Centers for Disease Control and Prevention (CDC) estimated at least2 million people in the United States become infected with bacteria thatare resistant to antibiotics and at least 23,000 people die each year asa direct result of these infections. (Antibiotic Resistance Threats inthe United States, 2013, Centers for Disease Control and Prevention,Atlanta, Ga., USA 2013). The CDC report classified three microorganismswith an antibiotic resistance threat level of urgent in the UnitedStates and twelve microorganisms with an antibiotic resistance threatlevel of serious. Specifically, Clostridium difficile,Carbapenem-resistant Enterobacteriacaeae (CRE) and drug resistantNeisseria gonorrhoeae (cephalosporin resistance) are classified by theCDC as urgent because they require urgent public health attention toidentify infections and to limit transmission. Of these, the CDC states“Clostridium difficile is the most frequent etiologic agent forhealth-care-associated diarrhea. In one hospital, 30% of adults whodeveloped health-care-associated diarrhea were positive for C.difficile. Risk factors for acquiring C. difficile-associated infectioninclude a) exposure to antibiotic therapy, particularly with beta-lactamagents; b) gastrointestinal procedures and surgery; c) advanced age; andd) indiscriminate use of antibiotics. Of all the measures that have beenused to prevent the spread of C. difficile-associated diarrhea, the mostsuccessful has been the restriction of the use of antimicrobial agents.”(Sehulster L, Centers for Disease Control and Prevention, Guidelines forenvironmental infection control in healthcare facilities. MMWR 2003;52(RR10); 1-42). C. difficile is an anaerobic, gram-positive bacteriumcapable of sporulating when environmental conditions no longer supportits continued growth. The capacity to form spores enables the organismto persist in the environment (e.g., on dry surfaces or in soil) forextended periods of time. Environmental contamination by thismicroorganism is well known, especially direct exposure to contaminatedpatient-care items and high-touch surfaces in patients' bathrooms havebeen implicated as sources of infection. The CDC stated, “More needs tobe done to prevent C. difficile infections (CDIs). Major reductions willrequire antibiotic stewardship along with infection control applied tonursing homes and ambulatory-care settings as well as hospitals. Statehealth departments and partner organizations have shown leadership inpreventing CDIs in hospitals and can prevent more CDIs by extendingtheir programs to cover other health-care settings.” (CDC, Vital Signs;Preventing Clostridium difficile infections, MMWR 2012; 61-157-62).Because C. difficile spores resist killing by usual hospitaldisinfectants, an Environmental Protection Agency-registereddisinfectant with a C. difficile sporicidal label claim should be usedto augment thorough physical cleaning.

Twelve serious antibiotic-resistant threats identified in the CDC reportinclude: multidrug-resistant Acinetobacter, Drug-resistantCampylobacter, Fluconazole-resistant Candida (fungus), Extended spectrumβ-lactamase producing Enterobacteriacaea (ESBLs), Vancomycin-resistantEnterococcus (VRE), Multidrug-resistant Psuedomonas Aeruginosa,Drug-resistant Non-typhoidal Salmonella, Drug-resistant SalmonellaTyphi, Drug-resistant Shigella, Methicillin-resistant Staphylococcusauereus (MRSA), Drug-resistant Steprtococcus pneumonia, Drug-resistanttuberculosis (MDR and XDR) (Antibiotic Resistance Threats in the UnitedStates, 2013, Centers for Disease Control and Prevention, Atlanta, Ga.,USA 2013). Of the twelve serious antibiotic-resistant threats identifiedin the CDC report, Methicillin-resistant Staphylococcus aureus (MRSA) isthe most frequently identified antimicrobial drug-resistant pathogen inU.S. hospitals. MRSA was one of the first pathogens to developresistance, first detected the United Kingdom in 1961. In 1999, MRSA wasresponsible for 37% of fatal cases of sepsis in the UK. Additionally,half of all S. aureus infections in the U.S. are resistant topenicillin, methicillin, tetracycline and erythromycin, leaving onlyvancomycin as an effective agent against S. aureus; however, strainswith intermediate levels of resistance, termed glycopeptide-intermediateStaphylococcus aureus (GISA) or vancomycin-intermediate Staphylococcusaureus (VISA), began appearing in the late 1990s and oxazolidinone,(linezolid) resistance in S. aureus was reported in 2001. Additionally,community-acquired MRSA (CA-MRSA) has now emerged as an epidemic that isresponsible for rapidly progressive, fatal diseases, includingnecrotizing pneumonia, sepsis, and necrotizing fasciitis. Outbreaks ofCA-MRSA infections have been reported in correctional facilities, amongathletic teams and military recruits, and in nurseries.

In addition to resistance, current antibiotics also have a limited usedue to allergic reactions in many patients (Romano A, Caubet J C.Antibiotic allergies in children and adults: from clinical symptoms toskin testing diagnosis. J Allergy Clin Immunol Pract. 2014January-February; 2(1):3-12).

Not only does resistance and allergic reactions to current antimicrobialagents result in increased patient morbidity and even mortality, butineffectiveness of current antimicrobial agents is also a major expenseto society. Surgical infections are costly not only because of cost oftreatment, including potentially hospitalization, but also the loss ofproductive work. This is exemplified in treatment of an infected totalknee replacement. The relative incidence of operative infections wasreported as 2.0% and 2.4% following total knee surgery. The most commoncause of revision total knee surgery (25.2%) is infection. (Bozic K J etal. The epidemiology of revision total knee arthroplasty in the UnitedStates. Clin Orthop Relat Res. 2010 January; 468(1):45-51). The annualcost of infected revisions to U.S. hospitals increased from $320 millionto $566 million during the study period and was projected to exceed$1.62 billion by 2020. (Kurtz S M, et al Economic burden ofperiprosthetic joint infection in the United States. J Arthroplasty.2012 September; 27(8 Suppl) The average cost of the surgical revision ofan infected total knee replacement was $116,383 in the years 2001through 2007. (Kapadia B H, et al. The Economic Impact of PeriprostheticInfections Following Total Knee Arthroplasty at a SpecializedTertiary-Care Center. J Arthroplasty. 2013 Oct. 17).

There is also a high cost to prophylactic antibiotic treatment prior toand around the time of surgery. (Chaweewannakom U. et al., Cost analysisof peri-operative antibiotic administration in total knee arthroplasty.J Med Assoc Thai. 2012 October; 95 Suppl 10:S42-7; Hebert C K, et al.,Cost of treating an infected total knee replacement. Clin Orthop RelatRes. 1996 October; (331):140-5). Furthermore, these costs may not becovered by government insurance in the U.S. especially with the largepersonal deductibles people have chosen under the Affordable Care Act.This problem already exists in Germany. This is a burden on the patientif they pay. If they do not pay then the cost is shifted to the doctorand the hospital. (Haenle M. et al. Economic impact of infected totalknee arthroplasty. Scientific World Journal. 2012; 2012:1 96515).

Treatment of the failed infected total joint may include repeat surgery,removal of the implant, insertion of an antibiotic impregnated spacer,hospitalization, therapy and return at later date to remove spacer andredo the total joint with hospitalization and long term antibiotics.(Garg P, et al. Antibiotic-impregnated articulating cement spacer forinfected total knee arthroplasty. Indian J Orthop. 2011 November;45(6):535-540).

Fungal infections also are problematic and have become less susceptibleto current antimicrobial agents. In hospitalized patients, fungalinfections are the fourth common cause of blood stream infection.Candida albicans is the major fungal pathogen of humans. (Warren, N G,American Society for Microbiology; 1995. 723; Bachmann, S P,Antimicrobial Agents Chemother, 2002; 46: 3591). It has been reportedthat mortality rate of patients with catheter related candidemiaapproaches 40%. (Fux, C A, Trends Microbiol, 2005; 13(1): 34; andTampakakis, E., Eukaryot Cell, 2009; 8:732). Biofilms of C. albicans arecapable of holding other micro-organisms and more likely to beheterogeneous with other bacteria and fungi in the environment and onmedical devices. (E Tampakakis, A Y Peleg, E Mylonakis. Eukaryot Cell,2009; 8:732.) Moreover, biofilm cells are significantly less susceptibleto antimicrobial agents. As a result, drug therapy for an implantinfection may be futile, and often, the only solution is mechanicalremoval of the implant. (Melissa J J, et al, Antimicrob AgentsChemother. 2009; 53(6): 2638; and Anderson, J B, Nat Rev Microbiol,2005; 3(7): 547). Biofilm formation also plays an important role inoutbreaks of C. albicans related infections.

As a result, there is a continuing need for new antimicrobials andcompositions, which are effective in reducing or preventingmicroorganism growth. The new antimicrobials and compositions can have abroad spectrum of utility without a history of overuse or resistance.The new methods and compositions can be applicable to promoting healingof wounds. In addition, the antimicrobials and compositions would haveno known allergic manifestation. Further the new antimicrobials andcompositions would be cost effective. There is also a need forantimicrobial compositions that can be effectively used on varioussurfaces, including high-touch surfaces such as light switches andbathroom fixtures, medical devices, patient-care items, and the like toreduce microbial contamination.

SUMMARY OF THE INVENTION

In its various embodiments, the present invention provides generally forcompositions and methods for reducing or preventing microbial growth,which are particularly useful for treating and promoting the healing ofwounds. Specifically, the present invention provides methods andpharmaceutical and nutraceutical compositions that reduce orsubstantially eliminate microbes.

In one embodiment, a method of promoting healing of a wound in a mammalis provided; comprising administering an anthocyanin or an anthocyanidinto the mammal in need of such treatment a therapeutically effectiveamount of the anthocyanin or anthocyanidin composition or compoundwherein microbial growth is reduced and local growth hormone activity isoptimized.

In a further embodiment, a method of promoting healing of a wound in amammal is provided; comprising administering an anthocyanin metaboliteor an anthocyanidin metabolite to the mammal in need of such treatment atherapeutically effective amount of the metabolite or anthocyanidinmetabolite compositions wherein microbial growth is reduced and localgrowth hormone activity is optimized.

Further, the invention provides methods and compositions thateffectively deliver the compositions to the affected wound. The presentinvention provides compositions and methods for use in the treatment ofa variety of wound problems, including but not limited to, burns,pressure wounds, abrasions, diabetic wounds, and skin infections. Thepresent invention provides compositions and methods for use in thetreatment of a variety of infections, including those from bloodtransfusions. Preferably, the therapeutic compositions and compounds areadministered orally or topically; however, the therapeutic compositionsand compounds may be administered by any conventional route including,for example, oral, topical, buccal, injection, pulmonary, intravenous,inhalant, subcutaneous, sublingual, or transdermal. Those of skill inthe art can readily determine the various parameters and conditions forproducing these compositions or formulations without resort to undueexperimentation.

In one aspect, the present invention provides a pharmaceuticalcomposition for promoting wound healing, comprising: a) an anthocyanin;or b) anthocyanidin. By way of example, the anthocyanin can be selectedfrom cyanidin-3-glucoside or delphinidin-3-glucoside,cyanidin-3-galactoside, and pelargonidin-3-galactoside. Also by way ofexample, the anthocyanidins can be selected from cyanidin, delphinidin,pelargonidin, malvidin and petunidin.

In one aspect, for example, the present disclosure provides apharmaceutical composition comprising: a) an anthocyanin metabolite; orb) an anthocyanidin metabolite. In another aspect, the present inventionprovides for a pharmaceutical composition for promoting wound healing,comprising: a) an anthocyanin metabolite; or b) and anthocyanidinmetabolite. By way of example, metabolites can be selected fromprotocatechuic acid and 2,3,4 trihydroxybenzaldehyde. In yet anotheraspect, the present disclosure provides a pharmaceutical compositioncomprising a phytochemical. In yet another aspect, the presentdisclosure provides a pharmaceutical composition comprising apterostilbene formulation. In yet another aspect, the present disclosureprovides a pharmaceutical composition comprising: a) a metabolite of ananthocyanin metabolite; or b) a metabolite of an anthocyanidinmetabolite. By way of example, the metabolites are metabolites selectedfrom protocatechuic acid, 2,3,4 trihydroxybenzaldehyde.

Further by way of example, the pharmaceutical composition can comprise:a) an anthocyanin; or b) an anthocyanidin; and c) a pharmaceuticallyacceptable carrier. Further by way of example, the pharmaceuticalcomposition can comprise: a) an anthocyanin metabolite; or b) ananthocyanidin metabolite; and c) a pharmaceutically acceptable carrier.Further by way of example, the pharmaceutical composition can comprise:a) a pterostilbene formulation; and b) a pharmaceutically acceptablecarrier. By way of example, the pharmaceutically acceptable carrier canbe selected from, but not limited to, any carrier, diluent or excipientcompatible with the other ingredients of the composition.

Further by way of example, the pharmaceutical composition can comprise:a) an anthocyanin; and/or b) an anthocyanidin; and/or c) a pterostilbeneformulation; and/or c) a pharmaceutically acceptable carrier; and/or d)an acceptable delivery carrier. By way of example, the delivery carriercan be formulated and administered as known in the art, e.g., fortopical, oral, buccal, injection, intravenous, inhalant, subcutaneous,sublingual or transdermal Further, said topical delivery carrier may beformulated and administered to any surface, including but not limited toskin, bone, synovium, cartilage, and implants. By way of example, theacceptable delivery carrier can be selected from any dermal ortransdermal carrier compatible with the other ingredients of thecomposition. In some embodiments, the acceptable delivery carrier is abiodegradable microsphere or a slow release bioadsorbable material. Byway of example, the acceptable delivery carrier can be selected from50/50 D, L lactide/glycolide or 85/15 D, L lactide/glycolide, both ofwhich are amorphous physically and, therefore, are non-reactive whenused as a carrier in a composition that is delivered in or to the body.

In yet another aspect and in other embodiments, the anthocyanidinprovided in any recited composition is provided less than 200 mM and inother embodiments about 100 μM. In some embodiments, the anthocyanin oranthocyanidin provided in any recited composition or method of use isprovided in a range of between 10 to 200 mM. In other embodiments, theanthocyanin metabolite or anthocyanidin metabolite provided in anyrecited composition or method of use is provided in a range of between20 to 200 mM. In yet other embodiments, the anthocyanin, anthocyanidin,or metabolites thereof provided in any recited composition or method ofuse is provided in a range of between 20 to 100 mM. In yet otherembodiments, the anthocyanin, anthocyanidin, or metabolites thereofprovided in any recited composition or method of use is provided in arange of between 20 to 50 mM. In some embodiments, the metabolite isprovided in any recited composition less than 100 mM and in others at 25mM.

In one aspect, for example, the present disclosure provides for anantimicrobial composition comprising: a) an anthocyanin metabolite; orb) an anthocyanidin metabolite; or a phytochemical. By way of example,metabolites can be selected from protocatechuic acid, 2,3,4trihydroxybenzaldehyde and a phytochemical can be a pterostilbeneformulation. By way of example, the anthocyanin, anthocyanidin andmetabolites thereof can have broad spectrum activity againstdisease-causing microbes. In another aspect of the present invention amethod of treating a wound of an individual is provided, comprisingadministering any of the recited pharmaceutical compositions by topicalapplication, transdermal, buccal, oral, gavage, and injection orintravenous.

The present disclosure also provides for methods for reducingmicroorganisms on any surface or liquid, comprising contacting a surfaceor liquid with any of the recited compositions.

The present disclosure also provides for compositions and methods forreducing microorganisms in foods comprising contacting a food with anyof the recited compositions.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 provides the minimum, maximum and optimum pH for growth ofmicroorganisms. Acidic environments retard proliferation of variousbacteria. Anthocyanins, anthocyanidins and main metabolites are unstablerelative to basic pH; thus, anthocyanins, anthocyanidins and mainmetabolites thereof have the potential to lower the pH of wound tissueas well as any surfaces and act as bacteriocidal or bacteriostatic.

FIG. 2 is the metabolic pathway of cyanidin-3-glucoside (C3G) andincludes the chemical structures of cyanidin-3-glucoside and cyanidinand their metabolites.

FIG. 3 is the chemical structure of Protocatechuic acid (PCA), adihydroxybenzoic acid, a type of phenolic acid. It is a major metaboliteof antioxidant polyphenols found in certain plants, including green tea.

FIG. 4 compares concentrations of C3G and PCA to determine optimaleffective concentrations. Bacterial burdens for P. aeruginosa werecompared after treatment with C3G or PCA at 48 and 96 hours. Aconcentration of PCA 25 mM was effective to reduce the bioburden withstatistical significance at 48 hours. C3G at 100 and 200 mMconcentrations were effective at reducing the bioburden at 48 and 96hours.

FIG. 5 is a chart disclosing potential sources of PCA.

FIG. 6 is a table providing a summary of the effectiveness of certainanthocyanins, anthocyanidins and a metabolite, including bactericidal orbacteriostatic activity. During this test, the purity, concentrationsand molecular weight of these test substances (compounds) were known.The carrier was water and the dose was accurately calculated.Delphinidin limited growth against C. perfringens, S. aureus, and MRSA.Pelargonidin limited growth of P. acnes, C. perfingens, S. aureus, MRSA,and S. pyogenes. Cyanidin CI was effective against C. difficile, Cprefringens, S. aureus ATCH 6538, S. aureus (MRSA) ATCH 33591, S.mutans, and S. pyogenes. A proprietary formulation ofcyanidin-3-glucoside (approximately 28% C3G by weight) had limitedeffectiveness during this study (18-24 hours for aerobes; 48 hours foranaerobes (C. albacans and L. casei); however, this C3G formulation, waseffective against P. acnes, E. coli, MRSA, K. pneumoniae and P.aeruginosa. Protocathechuic acid (PCA), the main metabolite fromanthocyanins and anthocyanidins, was effective against all bacteriatested as well as C. albicans and K. pneumonia. Importantly for skinwound treatment, PCA was effective against S. aureus 6538 and 33591(MRSA) and P. aeruginosa. PCA was also effective on C. albicans, whichis important considering its ability to form biofilms and difficulty intreating C. albicans when existing with a catheter or implant.

FIG. 7 is a table summarizing in vitro test results of 2,4,6Trihydroxybenzaldehyde and demonstrating its ability to act as anantimicrobial, including as a bactericidal or bacteriostatic.Specifically, 2,4,6 Trihydroxybenzaldehyde was effective against E.coli, K. pneumonia, P. aeruginosa, S. aureus 6538 and 33591 (MRSA);further it was effective against a fungi, Aureobasidium pullulans, ATCC15233.

FIG. 8A is a photographic image illustrating in vitro test results of2,4,6 Trihydroxybenzaldehyde against P. aeruginosa.

FIG. 8B is a photographic image illustrating in vitro test results of2,4,6 Trihydroxybenzaldehyde against S. aureus 33591 (MRSA).

FIG. 8C is a photographic image illustrating in vitro test results of2,4,6 Trihydroxybenzaldehyde against P. acnes.

FIG. 9A shows the results of a rodent back skin tape study whereconcentrations of PCA and C3G in a vehicle of water were utilized todetermine effectiveness against P. aeruginosa skin infections.

FIG. 9B shows the results of rodent back skin tape study whereconcentrations of PCA and C3G in a vehicle of water were utilized todetermine effectiveness against P. aeruginosa skin infections.

FIG. 10A shows the results of an additional rodent back skin tape studyto determine effective dosages of PCA and C3G in a vehicle of water thatwould be bactericidal for P. aeruginosa.

FIG. 10B shows the results of an additional rodent back skin tape studyto determine effective dosages of PCA and C3G in a vehicle of water thatwould be bactericidal for P. aeruginosa.

FIG. 10C shows the results of an additional rodent back skin tape studyto determine effective dosages of PCA and C3G in a vehicle of water thatwould be bactericidal for P. aeruginosa.

FIG. 11A shows the results of a rodent back skin study to determine theeffects of PCA and C3G on the local growth hormones in untreated skinwounds of rodents. A concentration of 25 mM PCA increased local growthhormone levels of IGF-1 at the site of the untreated skin wound.

FIG. 11B shows the results of a rodent back skin study to determine theeffects of PCA and C3G on the local growth hormones in untreated skinwounds of rodents. A concentration of 25 mM PCA increased local growthhormone levels of TGF-Beta at the site of the untreated skin wound.

FIG. 11C shows the results of a rodent back skin study to determine theeffects of PCA and C3G on the local growth hormones in untreated skinwounds of rodents. A concentration of 25 mM PCA increased local growthhormone levels of EGF at the site of the untreated skin wound.

FIG. 12A is a photographic image of rodents treated with a topicalsolution of C3G (28%); at an acidic pH, this solution maintains a purpleor red color and quickly metabolized at elevated pH levels, the C3Gchanges to a pink or even clear color. In mouse model experiments,however, as observed in the image, the purple color of C3G remained onthe rodent wound surface scar, thus indicating the pH remained acidic onthe wound surface. The C3G material on the surface was confirmed bysubsequent histology.

FIG. 12B is a photographic image of tissue from a study utilizing thehomogenized wound tissue method used in this study, whereby the purplecolor indicates that the wound probably retained an acidic pH.

FIG. 13A shows the IGF-1 response to 25 mM PCA in various environments,including tape stripped, tape stripped and infected with P. aeruginosa,tape stripped and treated with PCA, and tape stripped infected with P.aeruginosa, and PCA treated.

FIG. 13B shows the TGF-β response to 25 mM PCA in various environments,including tape stripped, tape stripped and infected with P. aeruginosa,tape stripped and treated with PCA, and tape stripped infected with P.aeruginosa, and PCA treated.

FIG. 13C shows the EGF response to 25 mM PCA in various environments,including tape stripped, tape stripped and infected with P. aeruginosa,tape stripped and treated with PCA, and tape stripped infected with P.aeruginosa, and PCA treated.

FIG. 14 is a photographic image of a cross section of rodent skin.

FIG. 15 is a photographic image of a cross section of rodent skin.

FIG. 16 is a photographic image of a cross section of rodent skin.

FIG. 17 is a photographic image of a cross section of rodent skin.

FIG. 18 is a photographic image of a cross section of rodent skin.

FIG. 19 is a photographic image of a cross section of rodent skin.

FIG. 20 is a photographic image of a cross section of rodent skin.

FIG. 21 is a photographic image of a cross section of rodent skin.

FIG. 22 is a photographic image of a cross section of rodent skin.

FIG. 23 is a photographic image of a cross section of rodent skin.

FIG. 24 is a photographic image of a cross section of rodent skin.

FIG. 25 is a photographic image of a cross section of rodent skin.

FIG. 26 is a photographic image of a cross section of rodent skin.

FIG. 27 is a photographic image of a cross section of rodent skin.

FIG. 28 is a photographic image of a cross section of rodent skin.

FIG. 29 is a photographic image of a cross section of rodent skin.

FIG. 30 is a photographic image of a cross section of rodent skin.

FIG. 31 is a photographic image of a cross section of rodent skin.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless otherwise indicated, all technical and scientific terms usedherein shall have the same meaning as is commonly understood by one ofordinary skill in the art to which the disclosed subject matter belongs.Unless otherwise indicated, the following definitions are applicable tothis disclosure. All publications referred to throughout the disclosureare incorporated by reference in their entirety. To the extent anydefinition or usage provided by any document incorporated herein byreference conflicts with the definition or usage provided herein, thedefinition or usage provided herein controls.

As used in the specification and claims, the singular forms “a,” “an,”and “the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “a composition” includesmixtures or combinations of two or more such compositions.

Throughout the specification and claims, the word “comprise” andvariations of the word, such as “comprising” and “comprises,” means“including but not limited to,” and is not intended to exclude, forexample, other components, extracts, additives, or steps. It is alsocontemplated that embodiments described as “comprising” components, theinvention also includes those same inventions as embodiments “consistingof” or “consisting essentially of.”

Ranges can be expressed herein as “approximately” or from “about” oneparticular value, and/or to “about” another particular value. When sucha range is expressed, another embodiment includes from the oneparticular value and/or to the other particular value.

A weight percent of a reagent, component, or compound unlessspecifically stated to the contrary, is based on the total weight of thereagent, component, composition or formulation in which the reagent,component, or compound is included, according to its usual definition.

By “reduce” or other forms of the word, such as “reducing” or“reduction,” is meant decrease or lower a characteristic (e.g.,inflammation, growth or viability of microorganisms).

By “promote” or other forms of the word, such as “promoting,” is meantto induce a particular event or characteristic, or delay the developmentor progression of a particular event or characteristic, or to minimizethe chances that a particular event or characteristic will occur.

“Treat” or other forms of the word, such as “treating,” “treatment” or“treated,” is used here to mean to administer a composition or toperform a method in order to induce, reduce, eliminate, and prevent acharacteristic (e.g., inflammation, growth or viability of microbes). Itis generally understood that treating involves providing an effectiveamount of the composition to the mammal or surface for treatment.

The term “vehicle” or “vehicle carrier” as used herein refers to meanthe manner in which the reagents or compositions may be delivered,including as a liquid, salve, soap, foam, cream, solution, gel, spray,powder, wipes, antibacterial treatments, wipes and the like.

The term “wound” or “wound associated condition” as used herein refersto a medical condition when the integrity of any tissue is compromised(e.g., burns, skin breaks, bone breaks, muscle tears, punctures,surgical incision sites, microdermabrasion site, skin graft site,). Awound may be caused by any act, infectious disease, underlyingcondition, fall, or surgical procedure. A wound may be chronic, such asskin ulcers caused by diabetes mellitus, or acute, such as a cut orpuncture from a sharp object, an animal bite or a gunshot.

The term “growth factors” or “local growth factors” include but are notlimited to, fibroblast growth factor (FGF), FGF-1, FGF-2, FGF-4,platelet-derived growth factor (PDGF), insulin-binding growth factor(IGF), IGF-1, IGF-2, epidermal growth factor (EGF), transforming growthfactor (TGF), TGF-β, TGF-α, and collagen growth factors, and/orbiologically active derivatives of these growth factors.

By “bactericidal” or “antimicrobial” is meant the ability to effect(e.g., eliminate, inhibit decrease, or prevent) microorganism growth,viability, and/or survival at any concentration.

By “bacteriostatic” is meant the ability to effect (e.g., stabilize orprevent future growth or prevent new growth) microorganism growth at anyconcentration. A bacteriostatic compound, agent or reagent does noteliminate or kill the bacteria.

By “additive” or “food additive” is meant to the use as a component ofany food (including any substance intended to sue in producingmanufacturing, packing, processing, preparing, treating, packaging,transporting, or holding food).

By “antiseptic” is meant an antimicrobial reagent or composition that isapplied to any surface, including skin or tissue, to effect (e.g.,eliminate, inhibit, decrease or prevent) microorganism growth,viability, and/or survival.

By “disinfect” or other forms of the word, such as “disinfectant” or“disinfecting,” is meant decrease or lower a characteristic (e.g.,eliminate, reduce, inhibit, decrease, or prevent) microorganism growth,viability or survival at any concentration. It is generally understoodthat disinfect involves providing an effective amount of the compositionto any surface, but particularly hard surfaces.

By “sanitize” or other forms of the word, such as “sanitizer” or“sanitizing,” is meant decrease or lower a characteristic (e.g.,eliminate, reduce, inhibit, decrease, or prevent) microorganism growth,viability or survival at any concentration. It is generally understoodthat sanitizing involves providing an effective amount of thecomposition to any surface. Further, it is generally understood thatsanitizing solutions and sanitizing components are those solutions thatmay be safely used on food-processing equipment and utensils and onother food-contacting conditions.

By “isolated” or “an isolate” as it refers to either the compounds orreagents described herein means not 100% by weight but ratherapproximately 95% to 97% of the compound or reagent by weight.

The term “alkyl” as used herein is a branched or unbranched hydrocarbongroup of 1 to 20 carbon atoms. Non limiting examples include methyl,ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl,heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl,eicosyl, tetracosyl, and the like. Further, the alkyl group can also besubstituted or unsubstituted.

The term “alkoxy” or “alkyoxy group” as used herein refers to a branchedor unbranched hydrocarbon chain having from 1 to 15 carbons and linkedto oxygens. Non-limiting examples include methoxy, ethoxy and the like.

The term “ProC3G™” (commercially available ChromaDex®, Inc. product)means a cyanidin 3-glucoside anthocyanin extracted from black rice andcontaining approximately 35% cyanidin 3-glucoside by weight with anadditional 5% other anthocyanins.

The term “pterostilbene” as used herein refers to a dimethyl etheranalog of resveratrol (trans-3,5-Dimethoxy-4′-hydroxy-E-stilbene or3′,5′-Dimethoxy-4-stilbenol). The term “pTeroPure®” (commerciallyavailable ChromaDex®, Inc. product) means a proprietary formulation ofpterostilbene.

The term “medicament” as used herein refers to any wound treatment,including but not limited to the group consisting of burn reliefmedications, anesthetic agents, wound cleansers, antiseptic agents, scarreducing agents, immunostimulating agents, antiviral agents,antikeratolytic agents, anti-inflammatory agents, antifungal agents,acne treating agents, sunscreen agents, dermatological agents,antihistamine agents, antibacterial agents, bioadhesive agents,inhibitors of prostaglandin synthesis, antioxidants, and mixturesthereof.

Unless stated to the contrary, a formula with chemical bonds shown onlyas solid lines and not as wedges or dashed lines contemplates eachpossible isomer.

The term “nutraceutical” as used herein refers to any food stuff,including a dietary supplement or fortified food, provided for potentialhealth and medical benefits.

Reference will now be made in detail to specific aspects of thedisclosed materials, compounds, compositions, and methods, examples ofwhich are illustrated in the following description and examples, and inthe figures and their descriptions.

The present invention provides methods, compositions and uses fortreating and promoting healing of a wound. More specifically, themethods and compositions described herein include the administration ofan anthocyanin or an anthocyanidin or metabolites thereof for promotingwound healing by reducing or preventing microbial growth and inducingthe activation and or optimization of growth hormones.

The methods and compositions are used for the treatment of mammals,including humans. As with humans, there is a need for new antimicrobialcompositions for the treatment of animals, including equine, canine andfeline, due to resistance or allergic reactions to current antimicrobialcompositions or agents. Therefore, the methods and compositionsdisclosed herein will be useful for the treatment and promotion of woundhealing in livestock as well as domestic pets and will havebroad-spectrum activity against microbes.

The present invention also provides methods, compositions and uses fortreating surfaces and liquids to reduce microbial growth. Morespecifically, the methods and compositions described herein includecontacting any surface with a composition comprising an anthocyanin oran anthocyanidin or metabolites thereof thereby reducing or preventingmicrobial growth on said surface. Further, the methods and compositionsdescribed herein include adding the composition comprising ananthocyanin or an anthocyanidin or metabolites thereof to liquid orfluid, including other sanitizing solutions and/or sanitizingcomponents, thereby reducing or preventing microbial growth on saidsurface. Further still, the methods and compositions described hereininclude adding the composition comprising an anthocyanin or ananthocyanidin or metabolites thereof to any other vehicle, including butnot limited to a powder, paste, cream foam, gel, wipes, other sanitizingcomponents and the like thereby reducing or preventing microbial growthon said surface.

Plants are rich in phytochemicals, including a variety of secondarymetabolites, such as tannins, terpenoids, alkaloids, and flavonoids.Many of these have been found in vitro to have antimicrobial properties.(see Dixon R A, Dey P M, Lamb C J. Phytoalexins: enzymology andmolecular biology. Adv Enzymol. 1983; 55:1-69). The mechanisms thoughtto be responsible for phenolic toxicity to microorganisms include enzymeinhibition by the oxidized compounds, possibly through reaction withsulfhydryl groups or through more nonspecific interactions with theproteins. (see Mason T L, Inactivation of red beet beta-glucan synthaseby native and oxidized phenolic compounds. Phytochemistry. 1987;26:2197-2202). Additionally, reports suggest that various phytochemicalsare effective against the biofilms. (see Tsuchiya H, Comparative studyon the antibacterial activity of phytochemical flavanones againstmethicillin-resistant Staphylococcus aureus. J Ethnopharmacol. 1996;50:27-34.) Probable targets in the microbial cell are surface-exposedadhesins, cell wall polypeptides, and membrane-bound enzymes. (see SternJ L, Phlorotannin-protein interactions. J Chem Ecol. 1996;22:1887-1899).

Studies and reports to date, however, have only focused upon extractsand compounds of which the exact ingredients are not known oridentified. Percentage of the primary or active molecules in theseextracts was not established in these reports. In addition, the activephytochemicals or molecules from these extracts have not been isolatedin such a manner that the concentrations are known or established letalone established a percentage by weight of what would is the activephytochemical (phytochemical molecule) or provides any information onthe purity of the remaining material.

Anthocyanins and anthocyanidins have been identified in the compoundsand extracts of the various phytochemicals. Further, anthocyanins andanthocyanidins are known to have the potential to be the pro-drug ofbioactive phenolic intermediates. (see Woodward G., Anthocyaninstability and recovery: Implications for the analysis of clinical andexperimental samples. J. Agric Food Chem. 2009, 57, 5271-5278).Consideration has now been given to the bioactive role of theanthocyanin and anthocyanidin metabolites, particularly protocatechuicacid. (see Galvan F., Protocatechuic acid: The missing human cyanidinsmetabolite. Mol Nutr. Food Res. 2008, 52, 266-267). Anthocyanins areabsorbed by gavage and previously it was thought that anthocyanins couldnot be absorbed in the intestines. When ingested, anthocyanins andanthocyanidins are unstable and are quickly metabolized into theirmetabolites. This instability and propensity to quickly degrade to theirmetabolites is a matter of interest when their antimicrobial nature isexamined. It has also been established that they are absorbed into theplasma. (Cao G, Prior R L. Anthocyanins are detected in human plasmaafter oral administration of an elderberry extract. Clin Chem 1999; 45:574-6.) For example, anthocyanins are rapidly metabolized; 75% of C3G isfound as PCA in the plasma within 30 minutes of ingestion. (Vitaglione,P. et al., Protocatechuic acid is the major human metabolite ofcyanidin-glucosides. J. Nutr. 2007, 137, 2043-2048).

Although the therapeutic potential for anthocyanins, anthocyanidins andtheir metabolites was considered in previous studies, these studies usedphytochemicals extracted from plants (plant extracts); indeed, thenature, number and concentrations of these phytochemicals were notidentified. Moreover, the purity of the material studied was unknown or,more importantly, not listed. Therefore the studies reporting use ofthese extracts rarely isolate or identify the active reagent from themany in the compound.

Extracts of phytochemicals have the likelihood of the presence of otherplant substances, including but not limited to other anthocyanindins,anthocyanins, metabolites and even cellulose and heavy metals. Sinceanthocyanidins and anthocyanidins are particularly unstable, themultiplicity of these substances in a compound substantially preventsidentification of the active component or compound. It may be the parentmolecule or the metabolite. It may be a combination working in synergy.

Thus, a central problem solved by the present invention is thedevelopment of pharmaceutical compositions for the treatment of woundscomprising the anthocyanins and anthocyanidins and their metabolites.Additionally, the anthocyanins and anthocyanidins or their metabolitesalone provide an effective method of promoting healing of wounds. Thesecompositions also have broad spectrum activity against a wide range ofmicrobes. These compositions, however, may also be used in combinationwith other wound treatments, including other antimicrobials. In certainembodiments, the additional antimicrobial is not sulfamethoxazole.

The identification of anthocyanins and anthocyanidins or combinations ofanthocyanins, anthocyanidins or their metabolites that are bactericidalor antimicrobial was determined by conducting in vitro testing.Anthocyanidins that were tested at 100 mM included delphenindin,pelargonindin, and cyanidin CI and cyanidin-3-glucoside. Protocatechuicacid and 2,4,6 trihydroxybenzaldehyde, the anthocyanidin metabolites,were also tested at the same concentrations. Referring to FIGS. 6-8,delphinidin limited growth against C. perfringens, S. aureus, and MRSA.Pelargonidin limited growth of P. acnes, C. perfingens, S. aureus, MRSA,and S. pyogenes. Cyanidin CI was effective against C. difficile, Cprefringens, S. aureus ATCH 6538, S. aureus (MRSA) ATCH 33591, S.mutans, and S. pyogenes. C3G (approximately 28% by weight) had limitedeffectiveness during this study (18-24 hours for aerobes; 48 hours foranaerobes (C. albacans and L. casei). This proprietary C3G formulation,however, was effective against P. acnes, E. coli, MRSA, K. pneumoniaeand P. aeruginosa. Protocathechuic acid (PCA), the main metabolite fromanthocyanins and anthocyanidines, was effective against all bacteriatested as well as C. albicans and K. pneumonia. Importantly for skinwound treatment, PCA was effective against S. aureus 6538 and 33591(MRSA) and P. aeruginosa. PCA was also effective on C. albicans, whichis important considering its ability to form biofilms and difficulty intreating C. albicans when existing with a catheter or implant. 2,4,6Trihydroxybenzaldehyde was effective against E. coli, K. pneumonia, P.aeruginosa, S. aureus 6538 and 33591 (MRSA); it also was effectiveagainst A. pullulans, ATCC 15233, a fungi.

While specific dosages of certain anthocyanins and anthocyanidins weredetermined to have the above mentioned effects against certain bacteria,in vivo testing were conducted to determine optimal dosages and toconfirm the ability of a topical application of these compounds to haveantimicrobial effect while prompting healing of a wound. It washypothesized that certain dose and interval topical application of awater soluble solution of PCA and/or C3G (28% of C3G by weight) atcertain concentrations based upon molecular weight would kill or reducethe bioburden of Pseudomonas aeruginosa while healing the wound asevidence by optimization of the local growth hormones and confirmed byhistological evidence. Referring to FIG. 9, a decrease in bacterialburden in the skin at 96 hours days was noted (CFU means colony formingunits). A concentration of 50 mM of PCA was found to be most effective;higher concentrations of PCA were not as effective at decreasingbacterial burdens. The most effective concentration of C3G was 100 mM.Importantly, histological evaluations of skin samples from the studyconfirmed healing at 48 and 96 hours with proliferation ofparafollicular cells and migration to cover the skin surface. There wasminimal inflammation in the dermis. There was collagen proliferation inthe dermis. In this application, in some embodiments, anthocyanins oranthocyanidins and metabolites thereof are provided in concentrations ofabout 10 to 200 mM. In other embodiments, anthocyanins, oranthocyanidins, or metabolites thereof are provided in any recitedcomposition or method of use in a range of between 20 to 200 mM. In yetother embodiments, the anthocyanin, anthocyanidin, or metabolitesthereof provided in any recited composition or method of use is providedin a range of between 20 to 100 mM. In yet other embodiments, theanthocyanin, anthocyanidin, or metabolites thereof provided in anyrecited composition or method of use is provided in a range of between20 to 50 mM. In a preferred embodiment, anthocyanins or anthocyanidinsand or their metabolites are provided in concentrations of about 100 mMor less.

As provided in FIG. 1, bacteria have a range of pH at which growth isoptimized, and most bacteria are more viable at basic pH ranges.Generally, anthocyanins, anthocyanidins and their metabolites also havean acidic pH and have the potential to have bactericidal orbacteriostatic modes of action. Because C3G and PCA reagents have anacidic pH, their bactericidal or bacteriostatic mode of action is bydirect contact with the bacteria.

Anthocyanins and anthocyanidins were further studied to determineeffects on wound healing, including whether they had any effect on theoptimization of local growth hormone activity at the wound site alongwith other supporting histological evidence of promoting healing.

Local growth hormones are important substances in the control of woundhealing. Equally as important, however, is to optimize the amount ofthese hormones desirable for promoting wound healing while avoiding scarformation and keloids.

Examples of common local growth hormones related to skin wound healinginclude Epidermal growth factor (EGF), Insulin-like growth factor-1(IGF-1) and Transforming Growth Factor-Beta (TGF-β). Epidermal growthfactor or EGF is a growth factor that stimulates cell growth,proliferation, and differentiation by binding to its receptor EGFR.IGF-1 is important in skin repair by stimulating keratinocyteproliferation and migration as well as collagen production byfibroblasts. Its expression is important during wound healing such thatretarded healing has been correlated with reduced IGF-1 levels. Whilelocal administration of IGF-1 to wound sites enhanced wound closure andstimulated granulation tissue formation, increased IGF-1 receptorexpression was reported in chronic wounds and in hypertrophic scars.Additionally, IGF-1 stimulation was associated with increased invasivecapacity of keloid fibroblasts. Systemic delivery of IGF-1 also causedhyperglycemia, electrolyte imbalance, and edema. Therefore it isdesirable to have slightly elevated but not over elevated IGF-1 by atreatment modality. TGF-β also is important in skin would healing;however, it is considered a pro-fibrotic growth factor and increasedlevels of TGF-β or prolonged presence has been identified as causinghypertrophic scaring.

Referring to FIGS. 11-13, tests were performed on rodent skin to explorethe effects of PCA on the local growth hormones in rodent skin. Aconcentration of 25 mM PCA increased local growth hormone levels at thesite of the untreated skin wound. In particular, FIG. 11 demonstratesthat a single reagent or compound would optimize local growth hormonesto promote healing without scarring. Approximately 25 mM PCA was theoptimal reagent and dose. As demonstrated in FIG. 13, optimization ispossible using the compositions of the present invention. In FIG. 13,all three local growth hormones were lowered in the simulated clinicalpathological environment (stripped and infected); however, the loweringof these hormones was not to the extent of absences. Hence, thenecessary IGF-1 is still above the controls in this environment;however, the scar forming properties of the other two hormones have beenmarkedly reduced. Therefore, optimization of local growth hormones isachieved. In FIG. 13, the optional concentration of PCA was confirmed as25 mM PCA in this situation and environment, meaning local growthhormone growth levels were optimized at this dosage such that IGF-1 asmoderately elevated while TGF-β and EGF levels were decreased. This isimportant to promote wound healing while preventing potential scarring.

The therapeutic effective dose may vary depending on a wide variety offactors. For instance, the dose may vary depending on the formulation,method of application of the therapeutic reagent or combination withother reagents, or compositions, compounds or combination ofcompositions or compounds to the wound.

Methods (General)

According to one aspect of this invention, there is provided anantimicrobial composition and a method of promoting wound healing byreducing microbial growth. A method of promoting healing of a wound in amammal is provided, comprising administering an anthocyanin or ananthocyanidin to the mammal in need of such treatment a therapeuticallyeffective amount of the anthocyanin or anthocyanidin compound whereinmicrobial growth is prevented or reduced and local growth hormoneactivity is optimized.

In a further embodiment, a method of promoting healing of a wound in amammal is provided; comprising administering an anthocyanin metaboliteor an anthocyanidin metabolite to the mammal in need of such treatment atherapeutically effective amount of the metabolite or an anthocyanidinmetabolite compound wherein microbial growth is reduced and local growthhormone activity is optimized.

In yet another aspect of the present invention, a method of treating P.acnes in a patient in need is provided, comprising administering ananthocyanin metabolite to the patient in need of treatment in antherapeutically effective amount of protocatechuic acid wherein growthof P. acnes is reduced.

In another aspect of the present invention, a method of prophylacticallytreating a preoperative skin incision site is provided, comprisingadministering an anthocyanin, an anthocyanidin and/or a metabolite to apatient in need of such treatment an effective amount of the anthocyaninor anthocyanidin compound wherein microbial growth is prevented orreduced.

In another aspect of the present invention, a method of disinfecting asurface comprising contacting said surface with an anthocyanin, ananthocyanidin and/or a metabolite thereof in an effective amount of theanthocyanin, anthocyanidin and/or metabolite compound wherein microbialgrowth is prevented, reduced or eliminated and, further, where themicrobial growth that is reduced is methicillin resistant staphylococcusaureus (MRSA).

In another aspect of the present invention, a method of disinfecting asurface is provided comprising contacting said surface with ananthocyanin, an anthocyanidin and/or a metabolite thereof or combinationthereof in an effective amount of the anthocyanin, anthocyanidin and/ormetabolite or combination thereof wherein microbial growth is prevented,reduced or eliminated and, further, where the microbial growth that isreduced may be an endogenous or exogenous source, including but notlimited to P. acnes, S. aureus, P. aeruginosa, E. coli, S. epidermidis,S. pneumonia, and Streptococcus species.

In another aspect of the present invention, a method of post-operativetreating a post-operative skin site is provided, comprisingadministering an anthocyanin, an anthocyanidin and/or a metabolite to apost-operative skin site, such as a skin graft, skin graft donor site, amicrodermabrasion site, or a surgical incision site, in an effectiveamount of the anthocyanin, anthocyanidin and/or metabolite compoundwherein microbial growth is prevented, reduced or eliminated and localgrowth hormone production is optimized.

In yet another aspect of the present invention, a method ofpost-operative treating a post-operative skin site is provided,comprising administering an anthocyanin, an anthocyanidin and/or ametabolite to a post-operative skin site, such as a skin graft, skingraft donor site, a microdermabrasion site, or a surgical incision site,in an effective amount of the anthocyanin, anthocyanidin and/ormetabolite compound wherein microbial growth is prevented, reduced oreliminated and local growth hormone production is optimized.

Compositions

Disclosed herein, in one aspect, are antimicrobial compositions. Thedisclosed antimicrobial reagents and compositions can be used toeliminate, reduce, and/or prevent microorganism growth, viability, orsurvival.

In some embodiments and aspects, the disclosed antimicrobial compositioncan be selected from the list of anthocyanins, anthocyanidins,metabolites of anthocyanin and anthocyanidin metabolites, or acombination thereof. By way of example, the anthocyanin can be selectedfrom cyanidin-3-glucoside or delphinidin-3-glucoside,cyanidin-3-galactoside, and pelargonidin-3-galactoside. Also by way ofexample, the anthocyanidins can be selected from cyanidin, delphinidin,pelargonidin, malvidin and petunidin. By way of example, metabolites canbe selected from protocatechuic acid, 2,3,4 trihydroxybenzaldehyde.

In yet another embodiment and aspect, the disclosed antimicrobialcomposition can be a phytochemical. By way of example, the phytochemicalis a pterostilbene formulation.

In one aspect, the present invention provides for a pharmaceuticalcomposition for promoting wound healing, comprising: a) an anthocyanin;or b) anthocyanidin. By way of example, the anthocyanin can be selectedfrom cyanidin-3-glucoside or delphinidin-3-glucoside,cyanidin-3-galactoside, and pelargonidin-3-galactoside. Also by way ofexample, the anthocyanidins can be selected from cyanidin, delphinidin,pelargonidin, malvidin and petunidin.

In one aspect, the present invention provides for a pharmaceuticalcomposition for promoting wound healing, comprising: a) a phytochemical.By way of example, the phytochemical is a pterostilbene formulation.

In one aspect, for example, the present disclosure provides for apharmaceutical composition comprising protocatechuic acid (PCA) wherebysaid composition reduces the growth of certain microbes, including P.acnes.

In one aspect, for example, the present disclosure provides for apharmaceutical composition comprising cyanidin-3-glucoside whereby saidcomposition reduces the growth of certain microbes, including H. pylori.

In one aspect, the present invention provides for a pharmaceuticalcomposition for treating a wound, comprising: a) an anthocyanin; b)anthocyanidin; or c) a metabolite of an anthocyanin or anthocyanidin inan effective amount whereby microbial growth is reduced. By way ofexample, the anthocyanin can be selected from cyanidin-3-glucoside ordelphinidin-3-glucoside, cyanidin-3-galactoside, andpelargonidin-3-galactoside. Also by way of example, the anthocyanidinscan be selected from cyanidin, delphinidin, pelargonidin, malvidin andpetunidin. By way of example, metabolites can be selected fromprotocatechuic acid (PCA) and 2,3,4 trihydroxybenzaldehyde.

In another aspect, the pharmaceutical composition of this invention totreat a wound generally can include a concentration of the anthocyaninsand anthocyanidins or metabolites thereof in a concentration of at least25 mM concentration, not including the carrier. In yet another example,the pharmaceutical composition of this invention to treat a wound caninclude a concentration of the anthocyanins and anthocyanidins ormetabolites thereof in a concentration of between 20 mM to 200 mMconcentration, not including the carrier. In yet other embodiments, thepharmaceutical composition of this invention to treat a wound caninclude a concentration of the anthocyanin, anthocyanidin, ormetabolites thereof provided in any recited composition or method of ina range of between 20 to 100 mM, not including the carrier. In anotherexample, the pharmaceutical composition of this invention to treat awound can include a concentration of the anthocyanin, anthocyanidin, ormetabolites thereof provided in any recited composition or method of ina range of between 20 to 500 mM, not including the carrier.

Further, in one example, in a pharmaceutical composition of thisinvention, PCA can be provided in a concentration approximately 50-100mM. Additionally, in one example, pterostilebene can be provided in aconcentration of approximately 35-65 mM. Further, in one example, PCAcan be provided in a concentration approximately 78 mM andpterostilebene at a concentration of approximately 40.6 mM to reducemicrobial growth or eliminate growth. Further, in one example, PCA andpsterostilbene can be provided in a combination in concentrationsprovided in this disclosure. In yet another example C3G would beprovided at a dosage of 131, 261 and 522 mg/kg.

In one aspect, the present invention provides for a pharmaceuticalcomposition for treating a wound, comprising: a) an anthocyanin; b)anthocyanidin; c) a metabolite of an anthocyanin or anthocyanidin; or d)a phytochemical in an effective amount whereby microbial growth isreduced. By way of example, the phytochemical is a pterostilbeneformulation.

The present disclosure also provides for a pharmaceutical compositioncomprising: a) an anthocyanin metabolite; or b) an anthocyanidinmetabolite. In another aspect, the present invention provides for apharmaceutical composition for promoting wound healing, comprising: a)an anthocyanin metabolite; or b) and anthocyanidin metabolite. In yetanother aspect, the present invention provides for a pharmaceuticalcomposition for promoting wound healing, comprising a phytochemical. Byway of example, metabolites can be selected from, protocatechuic acid,2,3,4 trihydroxybenzaldehyde. By way of example, the phytochemical canbe a pterostilbene formulation. In yet another aspect, the presentdisclosure provides for a pharmaceutical composition comprising: a) ametabolite of an anthocyanin metabolite; or b) a metabolite of ananthocyanidin metabolite. By way of example, the metabolites aremetabolites selected from protocatechuic acid, 2,3,4trihydroxybenzaldehyde. In yet another aspect, the present inventionprovides for a pharmaceutical composition comprising a phytochemical. Byway of example, the phytochemical can be a pterostilbene formulation.

The present disclosure provides for pharmaceutical compositions wherebythe anthocyanin, anthocyanidin, anthocyanin metabolite, anthocyanidinmetabolite, anthocyanin metabolite, or metabolites thereof, orphytochemical are isolated reagents.

The present disclosure also provides for routes of administration of thepharmaceutical compositions, including oral, injection, intravenous,topical, sublingual, buccal, inhalation, intradermal, subcutaneous, softtissue, and cutaneous.

Oral administration of the compositions of this disclosure, includingoral gavage, may include a liquid or semisolid form, tablet, pill,capsule, powder, or gel. Preferably, oral administration will be in aliquid composition. Compositions including a liquid pharmaceuticallyinert carrier such as water may be considered for oral administration.Other pharmaceutically compatible liquids or semisolids may also beused. The use of such liquids and semisolids is well known to those ofskill in the art.

Intravenous and injection administration will be in liquid form. Otherpharmaceutically compatible liquids or semisolids may also be used. Theuse of such liquids and semisolids is well known to those of skill inthe art.

Preferably, the composition is formulated as a topical composition. Morepreferable, the vehicle of the topical composition delivery is in theform of a liquid, salve, soap, spray, foam, cream, emollient, gel,ointment, balm or transdermal patch.

In addition to the components and administration of said compositionsdisclosed above, the compositions can be in the form of an aqueoussolution. The compositions disclosed herein can also be in the form of aliquid, gel, suspension, dispersion, solid, emulsion, aerosol, forexample, powders, tablets, capsules, pills, liquids, suspensions,dispersions or emulsions. Also, the compositions disclosed herein can bein the form suitable for dilutions. Similarly, the compositions can bein the form of a powder, cream, paste, gel or solid that can bereconstituted.

Other components can be present in the composition, if desired. Forexample, the antimicrobial composition can also include at least oneadditive selected independently from a carrier, a diluent, an adjuvant,a solubilizing agent, a suspending agent, a filler, a surfactant, asecondary antimicrobial agent, a preservative, a viscosity modifier, athixotropy modifier, a wetting agent, an emulsifier, or any combinationsthereof. For example, the disclosed antimicrobial composition canfurther comprise at least one surfactant selected from a cationicsurfactant, an anionic surfactant, a non-ionic surfactant, and anamphoteric surfactant. Additionally, the disclosed antimicrobial and/orpharmaceutical compositions may further comprise medicament is selectedfrom the group consisting of burn relief medications, anesthetic agents,wound cleansers, antiseptic agents, scar reducing agents,immunostimulating agents, antiviral agents, anti keratolytic agents,anti-inflammatory agents, antifungal agents, acne treating agents,sunscreen agents, dermatological agents, antihistamine agents,antibacterial agents, bioadhesive agents, inhibitors of prostaglandinsynthesis, antioxidants, and mixtures thereof.

Also, the disclosed antimicrobial compositions can optionally includeone or more additives such as carriers, adjuvants, solubilizing agents,suspending agents, diluents, surfactants, other antimicrobial agents,preservatives, fillers, wetting agents, antifoaming agents, emulsifiers,and additives designed to affect the viscosity or ability of thecomposition to adhere to and/or penetrate the wound.

In one embodiment, the disclosed antimicrobial compositions, includingthe selected active components, including the anthocyanins oranthocyanidins and metabolites thereof, are without causing significantundesirable biological effects or interacting in a deleterious mannerwith any of the other components of the composition in which it iscontained.

In one embodiment, the disclosed compositions, including the selectedactive components, including the anthocyanins or anthocyanidins andmetabolites thereof, are provided as a nutraceutical and provided as adietary supplement without causing significant undesirable biologicaleffects or interacting in a deleterious manner with any of the othercomponents of the foodstuff in which it is contained.

In one aspect, the antimicrobial composition provided herein, includinganthocyanins or anthocyanidins and metabolites thereof, are used inagricultural settings, including but not limited to nurseries,commercial farming, agricultural research facilities, residentialgardens and produce processing facilities, and are applied to plants andtrees to inhibit, reduce or substantially eliminate microbial bioburdenas well as many fungal bioburden on plants, trees, and surfaces thereof,including leaf surfaces.

In another embodiment, antiseptic compositions of the present inventionare formulated for use in liquids, solutions, gels, soaps, creams,powders salves and other preparations designed for topical use asantiseptic agents, sprays, foams, antibacterial treatments, wipes andthe like. In another embodiment, antiseptic compositions of the presentinvention are formulated as a hand antiseptic.

In yet another embodiment, antiseptic compositions of the presentinvention are used in industrial settings such as in water treatmentfacilities, including swimming pools or water treatment plants, foodpreparation, including but not limited to poultry and fish processingfacilities or produce handling and packaging settings to inhibit, reduceor substantially eliminate microbial bioburden, as well as many fungalbioburden. In addition to adding the antiseptic composition to a watersupply or water supply system, industrial equipment and surfaces may becontacted with, or soaked in, the antiseptic compositions of the presentinvention.

In yet another embodiment, sanitizing compositions of the presentinvention are formulated for use in liquids, solutions, gels, soaps, andother preparations designed for use as sanitizing agents, liquids,including sprays, foams, gels, soaps, sanitizing treatments, and thelike when used as a sanitizing solution, including but not limited to,use in food processing facilities, including food-processing equipmentand utensils, and on other food-contact articles.

In yet another embodiment, sanitizing compositions of the presentinvention use in food processing facilities, including food-processingequipment and utensils, and on other food-contact articles areformulated to include any components generally recognized as safe foruse in food processing facilities, including but not limited to, aqueoussolutions containing potassium, sodium or calcium hypochlorite, asolution of hydrogen peroxide, an aqueous solution containing potassiumiodide, sodium lauryl sulfate, sodium-toluenesulfonchloroamide,solutions containing dodecylbenzensulfonic acid, other acceptabledetergents and the like.

In one aspect, the one or more of the additives can be an agent that isacceptable when used in or on foods and beverages and which can beconsumed by a mammal (e.g., human, pet, livestock, etc.) along with theselected active components, including the anthocyanins or anthocyanidinsand metabolites thereof, without causing significant undesirablebiological effects or interacting in a deleterious manner with any ofthe other components of the composition in which it is contained.

In another aspect, the compositions of the present invention, includingthose compositions comprising: a) an anthocyanin; or b) ananthocyanidin; or c) a metabolite of an anthocyanin metabolite; or d) ametabolite of an anthocyanidin metabolite, or e) a combination thereof,are used in food processing, packing, manufacturing, handling,preparing, treating, transporting or holding as a food additive withoutcausing undesirable effects or interacting in a deleterious manner. Byway of example, protocatechuic acid can be used as an additive in meat,including the handling and processing, without causing undesirableeffects or interacting in a deleterious manner with the meat.

In yet another aspect, the compositions of the present invention,including those compositions comprising: a) an anthocyanin; or b) ananthocyanidin; or c) a metabolite of an anthocyanin metabolite; or d) ametabolite of an anthocyanidin metabolite, or e) a combination thereof,are used in food processing, including cold sterilization of foodcontainers, including bottles, without causing undesirable effects orinteracting in a deleterious manner.

In other examples, the antimicrobial compositions disclosed herein canfurther comprise a carrier. The term “carrier” means a compound,composition, substance, or structure that, when in combination with acompound or composition disclosed herein, facilitates preparation,administration, delivery, effectiveness, or any other feature of thecompound or composition. Examples of carriers include water, ethanol,polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like),vegetable oils, and suitable mixtures thereof. “Pharmaceuticallyacceptable carrier” means a compound, composition, substance, orstructure that is useful in neither preparing a pharmaceuticalcomposition which is generally safe, non-toxic, and neither biologicallynor otherwise undesirable and includes that which is acceptable forveterinary as well as human pharmaceutical use.

In a further example, the antimicrobial compositions disclosed hereincan also comprise adjuvants such as preserving, wetting, emulsifying,suspending agents, and dispensing agents. Prevention of the action ofother microorganisms can be accomplished by various antifungal agents,for example, parabens, chlorobutanol, phenol, and the like.

Suitable suspending agents can include, for example, ethoxylatedisostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters,microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agarand tragacanth, or mixtures of these substances, and the like.

The disclosed antimicrobial compositions can also comprise solubilizingagents and emulsifiers, as for example, ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl alcohol,benzyl benzoate, propyleneglycol, 1,3-butyleneglycol, dimethylformamide,oils, in particular, cottonseed oil, groundnut oil, corn germ oil, oliveoil, castor oil and sesame oil, glycerol, tetrahydrofur fury 1 alcohol,polyethyleneglycols and fatty acid esters of sorbitan or mixtures ofthese substances, and the like. The additives can be present in thedisclosed compositions in any amount for the individual anthocyanin oranthocyanidin compound components.

Compositions and Misc. Methods

This disclosure also provides for a method comprising contacting asurface with an effective amount of the antimicrobial composition. Bythe term “effective amount” of a composition as provided herein is meantan amount of a composition sufficient to provide the desired benefit,either bactericidal or bacteriostatic (e.g., reduction or prevention ofmicroorganism growth or survival). As disclosed herein, the exact amountrequired will vary from use to use depending on a variety of processingparameters, as understood by one of ordinary skill, such as the type ofsurface, the type of microorganism to be treated, the surface size, themode of deliver (e.g., aerosol, spraying or dipping), and the like.Determination of what constitutes an “effective amount” is made byroutine testing with known concentrations and adjusting thoseconcentrations as needed to obtain the desired benefit and can bedetermined by one of ordinary skill in the art using only routineexperimentation.

When the antimicrobial composition of this invention is applied to asurface to be treated, the antimicrobial composition generally caninclude a concentration of the anthocyanins and anthocyanidins of atleast 25 mM concentration, not including the carrier. Further, theisolated anthocyanins, anthocyanidins, or metabolites compounds thisinvention will be between 90%-97% by weight of the compound, and morepreferably, between 95%-98% by weight of the compound.

When the antimicrobial composition or compositions of this invention areapplied to a surface to be treated may be diluted for use as a sanitizeror as a preventive or prophylactically, and at greater concentrationsfor treatment.

EXAMPLES Example 1: Use of In Vitro Studies for AntimicrobialSusceptibility Testing of Anthocyanins, Anthocyanidins, or Metabolitesand Compounds Thereof

This example describes the method for testing the antimicrobialsusceptibility of anthocyanins, anthocyanidins, or metabolites andcompounds thereof. The Kirby-Bauer method of disc diffusion was used fortesting, following a standard set of procedures recommended by theNCCLS. In this methodology, a set of discs saturated with either testingcompounds or a control was placed on inoculated agar plates. The plateswere inoculated with organisms listed in the tables provided in FIG. 6,including C. difficile, P. acnes, C. prefringens, L. casei, C. albicans,E. coli, ATTC 8739 and ATCC 43895, S. aureus, S. mutans, S. pyogenes, P.aeruginosa and K. pneumonia. The control sample was amoxicillin, anantimicrobial with very effective broad spectrum antibiotic properties.Samples included delphinidin, pelargonidin, cyanidin CI, 28%cyanindin-3-glucoside (C3G), protocatechuic acid (PCA) and 2,4,6Trihydroxybenzaldehyde (2,4,6 THBA).

After 18, 24, or 48 hours of incubation, depending upon themicroorganism, each plate was examined. The diameters of the zones ofcomplete inhibition were measured, including the diameter of the disc.Zones were measured to the nearest millimeter, using sliding calipers.The size of the zones of inhibition was interpreted by referring toNCCLS standard. Results were interpreted as follows: NI was noinhibition of growth under the test sample, I was inhibition of growthunder the test sample, NZ indicated no zone of inhibition surroundingthe test sample, and CZ indicated a clear zone of inhibition surroundingthe sample and zone width in millimeters. See FIG. 6 for completeresults.

Results

Referring to FIG. 6, the testing samples had bactericidal andbacteriostatic activity against many of the organisms. Of note, P.acnes, an organism that is very difficult to treat, often requiringmultiple current antibiotics for effective treatment, was susceptible toboth C3G and PCA. Indeed, both of these test samples were bactericidalagainst P. acnes. Additionally, PCA was also effective againstStaphylococcus aureus ATCC 33591, known as Methacillin Resistant StaphAureus (MRSA).

PCA was also shown to have some effectiveness against Pseudomonasaeruginosa, a common pathogen in wounds, especially burns. Amoxicillin,the control sample, had no effect on P. aeruginosa. Similarly, Candidaalbicans, frequently a co pathogen in wounds, was susceptible to PCA.

In summary, the present invention provides advantages over the priorart, including providing anthocyanin, anthocyanidin, their metabolitesor combinations thereof to a wound to provide a reduction or eliminationof bacteria. It is contemplated that the invention will also find use inthe treatment of surfaces, including medical devices and medicalimplants, to reduce or eliminate bacteria.

Example 2: Use of Mouse Model to Determine Dose Levels and Intervals ofTest Samples

Methods:

Mice had back skin tape stripped and the stripped site (wound) wasinfected with P. aeruginosa (ACTA 9027). The test reagents were appliedtopically in an aqueous solution on the stripped site at two hours anddaily for four days.

Cyanidin 3-glucoside (C3G), an anthocyanin, and its main metabolite PCAwere formulated and tested at several doses. The aqueous carrier waswater. The C3G formulation included 50 mM, 100 mM and 200 mM doseconcentrations. Similarly, the PCA formulation included at 50, 100 and200 mM dose concentrations.

Results

Results were collected from the mice at day five. Both C3G and PCAdecreased the bacterial burden; however, none were statisticallysignificant. See FIG. 9a . There was a trend towards a decreasingconcentration of PCA, with 50 mM being the most effective. The mosteffective dose of C3G was 100 mM. It is contemplated that because C3Gdegrades to PCA in this environment, the test results may indicate thatC3G was not being tested alone, but rather was a combination of C3G andits metabolites, including a combination of C3G and PCA as the effectiveagents.

Example 3: Use of Mouse Model to Further Determine Effective Dose Levelsand Dose Intervals of Test Samples

Methods:

Mice had back skin tape stripped and the stripped site (wound) wasinfected with P. aeruginosa (ACTA 27853). The test reagents were appliedtopically in an aqueous solution on the stripped site at two hours anddaily on day 1, 2 and 3.

C3G, an anthocyanin and its main metabolite PCA were formulated andtested at several doses. The aqueous carrier was water. The C3Gformulation included 100 mM and 200 mM dose concentrations and the PCAformulation included 25 and 50 mM dose concentrations.

Results

Results were collected from the mice at day two and four. Both C3G andPCA decreased the bacterial burden at 48 and 96 hours. (See FIG. 9b )The most significant decrease of bacteria was observed at 25 mM of and100 and 200 mM of C3G. Although PCA at 25 mM reduced the bacterialburden at both time periods, its activity was statistically significantat 48 hours. C3G at both 100 mM and 200 mM significantly reduced thebacterial burden at 48 and 96 hours.

Example 4: Use of a Mouse Model for Wound Healing

Methods:

Mice were shaved but unstrapped and uninfected (normal rodent skin). Thetest reagents were applied topically in an aqueous solution on theunstripped site at two hours and daily on day 1, 2 and 3.

Testing reagents consisted of C3G and PCA formulated at one dose, 100 μMin an aqueous solution.

Results:

Referring to FIG. 10, there was little or no stimulation of IGF-1 andTGF-β at local levels observed at the 100 μM concentration of testingreagents. In fact, levels of EGF actually decreased below normal levels.There was observed a decrease of all three local growth hormones at 100uM of C3G. These results suggest that mice skin differs in response to adose that has been shown to stimulate human synovium to produce IGF-1.Thus, this low of a dose is not useful for rodents for this purpose.

Example 5: Use of Mouse Model to Determine Isolated Effect of 25 mMSolution of PCA in Various Environments

Methods:

Four different conditions were used: mice had back skin tape strippedand the stripped site (wound) was infected with P. aeruginosa; mice hadback skin stripped and were not infected, mice had taped stripped,infected and treated with PCA, mice were tape stripped, uninfected, andtreated with PCA. When used, the PCA test reagent was applied topicallyin an aqueous solution on the stripped site at two hours and 24 hours.

The testing reagents consisted of and PCA formulated at one dose, 25 mM,in an aqueous solution. Levels of IGF-1, TGF-β, and EGF levels in theskin tissue at 48 hours were measured by ELISA. There were two controlgroups; the stripped skin and the stripped skin and infected.

Results:

Referring to FIG. 13, the infected stripped skin showed the highestlevel with IGF-1 (statistically significant) and TGF-β. This isrepresentative of tissue response to injury and infection; similarly,the EGF response was very inconsistent compared to the other two growthhormones.

The EGF response levels were different than either IGF-1 or TGF-β. Theywere highest in the stripped and uninfected wound and lowest in thestripped, infected and treated wound. Therefore, the treatment optimizedthe amount of hormone production compared to the untreated infection.This is beneficial to limit scarring while promoting healing over thecontrols. Overall, PCA at 25 mM acts on stripped and infected mice skinand optimizes the IGF-1 production and optimizes the local growthhormones.

Example 6: Use of Mice to Establish Wound Promoting Effect ofCompositions

Method:

Fifteen rodents were used to establish the histological findings ofstripped skin, stripped and infected skin, and stripped, infected andtreated wound. There were two control groups and four experimentalgroups according to the following:

Control Group 1: three mice with only tape stripped wounds on the back.These mice were not infected or treated. The skin was harvested at timezero, 2 and 48 hours for histology examination.

Control Group 2: three had tape stripped wounds and infection. Tissuesubmitted at 2 and 48 hours for histological examination.

Experimental Groups: There were 4 experimental groups. In these groups,mice had skin stripped wounds and infection. Treatment varied by reagentand dosage. Testing reagents included PCA at 25 at 25 and 50 mM and C3Gat 100 and 200 mM.

Pseudomonas aeruginosa (ATCC 27853) procured from American Type CultureCollection, Manassas, Va. was used to infect the experimental groups ofmice. The organism was grown overnight at 37° C. at ambient atmospheretrypticase soy agar plates supplemented with 5% sheep blood cells. Theculture will be aseptically swabbed and transferred to tubes oftrypticase soy broth. The optical density will be determined at 600 nm.The cultures will be diluted to provide an inoculum of approximately 9.0log₁₀ CFU per mouse in a volume of 100 μL. Inoculum count was estimatedbefore inoculation by optical density and confirmed after inoculation bydilution and back count.

The testing reagents were topically applied at 2 and 24 hours with 100uL of fluid spread over the wound.

The following histological assessments were conducted:

Surface Cellularity: The histological assessment included the presenceor absence of the surface cellularity and the depth of the cells.

Dermis:

Thickness: The thickness of the dermal layer was observed.

Hair Follicles: The hair follicles and the layer of surrounding cellswere observed. Hair follicles presence is critically important to skinwound healing. (Gharzi A, Reynolds A J, Jahoda C A. Plasticity of hairfollicle dermal cells in wound healing and induction. Exp Dermatol. 2003April; 12 (2):126-36). The dermal sheath surrounding the hair folliclehas the progenitor cells for contributing fibroblasts for wound healing.(Johada C A, Reynolds A J. Hair follicle dermal sheath cells: unsungparticipants in wound healing. Lancet. 2001 Oct. 27; 358(9291):1445-8).

Vascularity: Vascularity was observed, but an assessment of angiogenesiswas not performed on the 48 hour material since new vascularity takesthree to twelve days to develop. (Busuioc C J, et al. Phases ofcutaneous angiogenesis process in experimental third-degree skin burns:histological and immunohistochemical study. Rom J Morphol Embryol. 2013;54(1):163-710.)

Inflammation: The presence of cellular infiltration was observed and itslocation.

Skin Thickness: The thickness of the skin was estimated related to theuninfected, untreated wound. This depth was estimated on the uniformhistology photomicrographs from the surface to the muscle layer.

Results:

The following results were observed in each group:

Control Group 1: Uninfected and Untreated.

Time Zero: (See FIGS. 14-15) At time zero following the wound strippingthere was cellular covering of the surface. The dermal layer was notthickened. The hair follicles have a single cellular lining. There wasminimal vascularity and no inflammation. The depth of the tissue wasconsidered zero for future bench mark. 0+

2 hours: (See FIGS. 16-17) At 2 hours following the wound stripping thesurface remained covered with cellularity. The dermal layer wasminimally thickened. The follicles and cellular lining was the same.There was minimal increase in vascularity and inflammation. The increasein the depth of the tissue was considered 0.5+.

48 hours: (See FIGS. 18-19) At 48 hours the wound stripped, uninfected,untreated specimens showed natural history response of surface cellularproliferation and thickness. The dermal layer was thickened. The hairfollicles were present with single layer cellular lining. Thevascularity was increased in amount compared to the 2 hour specimens.The inflammation was present throughout the dermis and muscle layer. Thethickness was considered 0.5+.

Control Group 2: Infected and Untreated.

2 hours: (See FIGS. 20-21) The histological assessment showed the woundstripped, infected, but untreated controls at 2 hours to have multiplecellular covering on surface. There was minimal thickening of the dermallayer. The hair follicles were abundant and had double layer cellularlining. There was minimal vascularity and no inflammation in thespecimens. The thickness was assigned 0.5+.

48 hours: (See FIGS. 22-23) At 48 hours the surface cellular coveringwas gone. The dermal layer had minimal thickening. The hair follicleswere present, with minimal cellularity lining. There was marked increasein vascularity and minimal inflammation in dermis layer. The depth wasconsidered 0.5+ compared to time zero.

Experimental Group PCA 25 mM

48 hours: (See FIGS. 24-25) The cellular covering of the surface wasabundant and multiple cell layers. The dermal layer was thickened. Thehair follicles were prominent with multiple cellular lining. There wascollagen proliferation between the epidermis and dermis. Additionally,there was moderate vascularity, but less than that seen in infecteduntreated group. There was abundant inflammation and it was greater thanwas seen in the PCA 50 dose. Thickness was assigned 2+.

Experimental Group PCA 50 mM

48 hours: (See FIGS. 26-27) The surface was covered with multiple layersof cells. The dermal layer was thicker. The hair follicles had doublelayer of cells. There was increased vascularity. Inflammation alsoincreased in the dermis and below the muscle layer. The tissue thicknesswas assigned 2+.

Experimental Group C3G 100 mM

48 Hours: (See FIGS. 28-29) There was multiple cellular covering of thesurface. The dye of the C3G was apparent on the skin surface indicatingit had not changed color due to pH nor completely degraded. The dermallayer was thicker. The hair follicle had single and double cellularlining. The vascularity was prominent. There was inflammation in thedermis and muscular layer and below. The thickness of the tissue wasassigned 2+.

Experimental Group C3G 200 mM

48 Hours: (See FIGS. 30-31) There was evidence of the C3G materialremaining on the skin surface. The surface cellular layer was multiplecells thick. The dermal layer was thickened. The hair follicles hadsingle and double cellular lining. The vascularity was increased. Therewas inflammation in the dermis and muscular layer. The thickness wasassigned 2+.

These results confirm that an anthocyanin (˜38% C-3-G as the source) andthe main metabolite of anthocyanins and anthocyanidins, protocatechuicacid (PCA) when applied topically at various calculated doses to thestripped skin wound of a rodent were bactericidal in 48 to 96 hours.There was a 10,000 fold kill of Pseudomonas aeruginosa in 48 hours withboth reagents and dose.

The results also show by histology a simultaneous healing of theexperimentally created wound in the same time frame. C-3-G and PCA intwo different doses stimulated tissue repair as evidence by histology.

Specifically, the experimental model provided evidence of a histologicalcontrast between the control and experimental groups. At 48 hours,Control Group 2 that was wound stripped and infected showed a clearcontrast to the uninfected Control Group 1. In the skin strippedinfected group there was loss of the epithelial cellular covering, nofollicular cellular proliferation, marked increase in vascularity andlittle inflammatory response. This histological condition provided clearcontrast to the treatment groups. All treatment groups by comparisonshowed healing response with multiple layer cellular proliferation onthe surface, multiple layer cellular proliferation along the hairfollicles, less vascularity, but an inflammatory cellular response inthe dermis and muscular levels. See FIGS. 14-31. PCA at a concentrationof 25 mM also showed collagen layer formation between the epidermis anddermis. (See FIGS. photos 24 and 25). This response is beneficial in theuse of anthocyanin and anthocyanidins and metabolites thereof as acosmetic agent to promote wound healing and improve skin health,including wrinkle reduction or removal. This method of use ofanthocyanin and anthocyanidin metabolites, and particularly PCA, isbased upon the two fold response; the collagen layer increase and theskin swelling that increased the depth of the skin.

What is claimed is:
 1. A method for reducing the growth of amicroorganism in a foodstuff, comprising: applying an antimicrobialcomposition to the foodstuff, the antimicrobial composition comprisingone or more of an anthocyanin metabolite or an anthocyanidin metabolitein an amount greater than or equal to 0.15% by weight selected from thegroup consisting of: protocatechuic acid, 2,4,6-trihydroxybenzaldehyde,and combinations thereof, and a nutraceutically acceptable carrier,wherein the anthocyanin metabolite or anthocyanidin metabolite inhibitsmicrobial growth of one or more of the following microorganisms:Bacillus cereus, Clostridium difficile, Clostridium sporogenes,Propionibacterium acnes, Clostridium perfringens, Escherichia coli,Klebsiella pneumoniae, Legionella, Lactobacillus casei, Micrococcusluteus, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcusmutans, Streptococcus pyogenes, Salmonella species, Salmonella typhi,Shigella species, Aspergillus fumigatus, Aureobasidium pullulans,Candida albicans, and Penicillium chrysogenum.
 2. The method accordingto claim 1, wherein the foodstuff is selected from a beverage, a dietarysupplement, a nutritionally fortified food, and a feed for animalconsumption.
 3. The method according to claim 1, wherein the one or moreof the anthocyanin metabolite or anthocyanidin metabolite is present inthe foodstuff in an amount from about 0.3% to about 40% by weight. 4.The method according to claim 1, wherein the one or more microorganismsincludes a second microorganism selected from: Bacillus cereus,Clostridium difficile, Clostridium sporogenes, Propionibacterlum acnes,Clostridium perfringens, Escherichia coli, Klebsiella pneumoniae,Legionella, Lactobacillus casei, Micrococcus luteus, Pseudomonasaeruginosa, Staphylococcus aureus, Streptococcus mutans, Streptococcuspyogenes, Salmonella species, Salmonella typhi, Shigella species,Aspergillus fumigatus, Aureobasidium pullulans, Candida albicans, andPenicillium chrysogenum.
 5. The method according to claim 1, wherein theone or more of the anthocyanin metabolite or anthocyanidin metabolite isPCA.
 6. The method according to claim 1, wherein the antimicrobialcomposition is used in producing, manufacturing, preparing, packaging,or transporting the foodstuff.
 7. The method according to claim 6,wherein the packaging is a container for a foodstuff.
 8. The methodaccording to claim 7, wherein the container is a bottle.
 9. The methodaccording to claim 1, wherein the antimicrobial composition is appliedonto the foodstuff.
 10. The method according to claim 1, wherein theantimicrobial composition is applied into the foodstuff.
 11. The methodaccording to claim 1, wherein the antimicrobial composition is appliedas an aerosol, a spray, a dip, or a combination thereof.
 12. The methodaccording to claim 1, wherein the antimicrobial composition furthercomprises an antifungal agent.
 13. An antimicrobial food additive forreducing growth of a microorganism in a foodstuff, comprising: an amountgreater than or equal to 0.15% by weight of one or more of ananthocyanin metabolite or an anthocyanidin metabolite selected from thegroup consisting of: protocatechuic acid, 2,4,6-trihydroxybenzaldehyde,and combinations thereof; and a nutraceutically acceptable carrier;wherein the anthocyanin metabolite or anthocyanidin metabolite inhibitsmicrobial growth of one or more of the following microorganisms:Bacillus cereus, Clostridium difficile, Clostridium sporogenes,Propionlbacterium acnes, Clostridium perfringens, Escherichia coli,Klebsiella pneumoniae, Legionella, Lactobacillus casei, Micrococcusluteus, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcusmutans, Streptococcus pyogenes, Salmonella species, Salmonella typhi,Shigella species, Aspergillus fumigatus, Aureobasidium pullulans,Candida albicans, and Penicillium chrysogenum when applied to the foodstuff.
 14. The antimicrobial food additive according to claim 13,wherein the foodstuff is selected from a beverage, a dietary supplement,a nutritionally fortified food, and a feed for animal consumption. 15.The antimicrobial food additive according to claim 13, wherein theconcentration of the one or more of the anthocyanin metabolite oranthocyanidin metabolite is present in the foodstuff in a range fromabout 0.3% to about 40% by weight.
 16. The antimicrobial food additiveaccording to claim 15, wherein the one or more of the anthocyaninmetabolite or anthocyanidin metabolite is protocatechuic acid.
 17. Afoodstuff, comprising: a food and one or more of an anthocyaninmetabolite or an anthocyanidin metabolite selected from the groupconsisting of protocatechuic acid, 2,4,6-trihydroxybenzaldehyde, andcombinations thereof; wherein the one or more of the anthocyaninmetabolite or anthocyanidin metabolite is present in the foodstuff in anamount greater than or equal to about 0.15% by weight; and wherein theamount is effective to inhibit microbial growth of one or more of thefollowing microorganisms: Bacillus cereus, Clostridium difficile,Clostridium sporogenes, Propionibacterium acnes, Clostridiumperfringens, Escherichia coli, Klebsiella pneumoniae, Legionella,Lactobacillus casei, Micrococcus luteus, Pseudomonas aeruginosa,Staphylococcus aureus, Streptococcus mutans, Streptococcus pyogenes,Salmonella species, Salmonella typhi, Shigella species, Aspergillusfumigatus, Aureobasidium pullulans, Candida albicans, and Penicilliumchrysogenum in the foodstuff.
 18. The foodstuff according to claim 17,wherein the one or more of the anthocyanin metabolite or anthocyanidinmetabolite is present in the foodstuff in an amount from about 0.3% toabout 40% by weight.
 19. The foodstuff according to claim 17, whereinthe foodstuff is in the form of a beverage, a dietary supplement, anutritionally fortified food, or a feed for animal consumption.
 20. Thefoodstuff according to claim 18, wherein the one or more of theanthocyanin metabolite or the anthocyanidin metabolite is PCA.
 21. Thefoodstuff according to claim 19, further comprising an antifungal agent.